EP1231277A2 - Biokompatibles Implantat zur in vivo Herstellung eines Neo-organs - Google Patents

Biokompatibles Implantat zur in vivo Herstellung eines Neo-organs Download PDF

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Publication number
EP1231277A2
EP1231277A2 EP02003797A EP02003797A EP1231277A2 EP 1231277 A2 EP1231277 A2 EP 1231277A2 EP 02003797 A EP02003797 A EP 02003797A EP 02003797 A EP02003797 A EP 02003797A EP 1231277 A2 EP1231277 A2 EP 1231277A2
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EP
European Patent Office
Prior art keywords
cells
implant
vector
promoter
collagen
Prior art date
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Withdrawn
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EP02003797A
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English (en)
French (fr)
Inventor
Philippe Moullier
Olivier Danos
Jean-Michel Heard
Nicolas Ferry
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Institut Pasteur
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Institut Pasteur
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Priority claimed from FR9304700A external-priority patent/FR2704236B1/fr
Priority claimed from FR9309185A external-priority patent/FR2708202B1/fr
Application filed by Institut Pasteur filed Critical Institut Pasteur
Publication of EP1231277A2 publication Critical patent/EP1231277A2/de
Withdrawn legal-status Critical Current

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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
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    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
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    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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Definitions

  • the invention relates to a biocompatible implant. for expression in humans or animals of specific substances, in particular for anchoring recombinant cells of the invention.
  • the present invention also relates to retroviral vectors for the preparation of recombinant cells capable of being implanted in vivo for therapeutic purposes.
  • the prior international patent application published under the number 92/15676 describes means for carrying out the expression of specific nucleotide sequences in vivo , for the therapeutic treatment of diseases resulting from a genetic anomaly.
  • This international application 92/15676 proposes to use fibroblasts genetically modified by a retroviral vector, with a view to implanting them in the connective tissue of the skin of the subject to be treated.
  • the nucleotide sequence whose expression is sought in this international application anterior is placed under the control of the sequence LTR (Long Terminal Repeat) of the retroviral vector and / or under the control of constitutive exogenous promoters or inducible, the LTR sequence of the retrovirus being nevertheless retained when the exogenous promoter is present.
  • LTR Long Terminal Repeat
  • the invention provides means for carrying out in vivo the expression of a selected nucleotide sequence, under conditions such that this expression is obtained over a period of several months, preferably more than 6 months, in such a way that the product of the expressed nucleotide sequence is present in sufficient quantity and under conditions suitable to produce a desired therapeutic effect in vivo .
  • the means defined in the present application make it possible to achieve the expression and the secretion of a protein, glycoprotein, or of a peptide having a biological interest, to authorize its therapeutic use in vivo .
  • the invention also relates to the use of biocompatible materials, as supports for the introduction in humans or animals of cells, for example recombinant cells of the invention defined below, from which wishes to produce a specific molecule, in particular therapeutic purposes.
  • the invention also relates to retroviral vectors having the capacity to infect cells and in particular eukaryotic cells as well as the recombinant cells comprising these vectors.
  • the recombinant cells of the invention can be administered, or implanted in a patient, and thus producing in vivo a protein or any expression product of a determined nucleotide sequence, inserted into a retroviral vector.
  • the inventors examined what were the parameters which, at different levels, would make it possible to express in vivo a determined nucleotide sequence, so as to improve the expression of the nucleotide sequence inserted into the vector in order to obtain a therapeutic effect, if necessary of long duration.
  • the exogenous nucleotide sequence we are looking for the expression is advantageously placed in the vector intended for cell infection, under control of a constitutive or inducible exogenous promoter, the LTR sequence internal to the retroviral vector must then be partially deleted. The deletion must be sufficient to affect transcription of mRNA.
  • the implant or neo-organ of the invention is obtained by the process which consists in assembling in in vitro of different elements in order to form an implant which is preferably introduced into the cavity peritoneal of the recipient. Other locations are usable such as the perirenal space, the dermis. In vivo, the implant gives rise to tissue neo-formed connective, vascularized, not modified during time.
  • the subject of the invention is therefore an implant or neo-organ characterized in that it comprises a support rigid biocompatible, particularly a support rigid PTFE or biological, allowing the biological anchoring of cells brought into contact, beforehand or not with capable constituents to induce and / or promote their inclusion within of a gel-forming matrix, said cells being chosen for their ability to express and secrete naturally or after recombination a compound determined, for example a compound of interest therapeutic.
  • a support rigid biocompatible particularly a support rigid PTFE or biological
  • biological anchorage means that the cells in the implant can get attach to the surface of the biocompatible support or, in in some cases, get inside this support.
  • This fixation of cells on the support is permitted in particular by the presence of the constituents capable of inducing and / or promoting the inclusion of cells within a matrix having the constitution of a gel.
  • gelation allows the organization of cells into a three-dimensional structure in an amorphous environment, not giving rise to prolonged inflammation in vivo .
  • the implant obtained is according to a first mode of realization of the invention consisting on the one hand of a biocompatible support, such as a support comprising a synthetic biocompatible material, especially polytetrafluoroethylene (PTFE) fibers or a support comprising a material of biological origin based on limestone, in particular based on calcium carbonate and preferably coral and secondly a gel possibly charged with cells expressing the substance of interest, including cells Recombinant.
  • a biocompatible support such as a support comprising a synthetic biocompatible material, especially polytetrafluoroethylene (PTFE) fibers or a support comprising a material of biological origin based on limestone, in particular based on calcium carbonate and preferably coral and secondly a gel possibly charged with cells expressing the substance of interest, including cells Recombinant.
  • a biocompatible support such as a support comprising a synthetic biocompatible material, especially polytetrafluoroethylene (PTFE) fibers or a support comprising a material of biological origin based on limestone, in particular
  • Implant comprising a biocompatible material synthetic.
  • the subject of the invention is an implant or neo-organ characterized in that it comprises cells expressing the substance of interest, in combination with polytetrafluoroethylene (PTFE) fibers and collagen.
  • PTFE polytetrafluoroethylene
  • the implant as described above further comprises a growth factor, for example bFGF (basic Fibroblast Growth Factor).
  • a growth factor for example bFGF (basic Fibroblast Growth Factor). This growth factor promotes vascularization of the neo-organ when it is implanted in vivo .
  • the neo-organ is introduced in the peritoneal cavity in a patient in whom we wish to obtain the expression of the sequence of exogenous nucleotides contained in the vector the invention.
  • This localization of the neo-organ is favorable to the development of its vascularity and to maintenance in an individualized form of this organ.
  • Implantation of the neo-organ including cells recombinants according to the invention can be permanent, or on the contrary transient, the neo-organ formed can in this case be removed after a given time implantation.
  • Implant comprising a biocompatible material of biological origin.
  • the material used is a high porosity coral, that is to say it has a porosity as described in the French patent No. 2,460,657. It may also be a powder of sufficient porosity to allow obtaining a solid framework for networking cell with or without a biological support such than collagen. In case the coral is replaced by collagen, crosslinked or not, the latter can also serve both as a support and as matrix promoting biological gelation for constitution of the cell network.
  • the high porosity coral used is spherical type.
  • the solid framework of the support of biological origin is capable of resorption in vivo .
  • the implant thus formed allows the formation in vivo of an individualized and stable neo-organ if necessary, in which the biological support is likely to be gradually absorbed, leaving room for a structure comprising a vascularized connective tissue of form relatively comparable that of the implant, this neo-organ however having, where appropriate, a volume reduced relative to the volume of the original implant.
  • a particularly preferred implant in the framework of the invention is such that the constituents capable of inducing and / or promoting gelation cells are for example uncrosslinked collagen or alginates.
  • collagen for example, type I, in particular to a concentration of 1.5 mg / ml.
  • a particular implant specific to the implementation of the invention is further characterized in that the cells are recombinant cells comprising genetic information foreign to their genome, capable of expressing this additional information in vivo and having the capacity to be tolerated immunologically by an organism to which they would be administered.
  • an implant according to the invention is characterized in that the cells are modified by a vector containing one or more nucleotide sequences exogenous, coding for an antigen or determinant antigenic or encoding a polypeptide or a glycoprotein, soluble in serum, for example for a polypeptide or a glycoprotein of interest therapeutic, in particular for a hormone, a protein or a structural glycoprotein or a protein or a metabolism glycoprotein or a protein or a viral glycoprotein or a protein with the characteristics of an antibody or fragment antibody.
  • the implant contains in in addition to one or more angiogenic factors, particular of bFGF, preferably incorporated during bringing the biocompatible support into contact with cells and the constituent capable of inducing and / or promote their gelation.
  • angiogenic factors particular of bFGF
  • the fixation of the angiogenic factor is favored by the presence of the gelling agent.
  • This agent contributes to the stable fixation of angiogenic factors with biological support of the implant.
  • the implant also contains heparin or a derivative heparin such as heparan sulfate or fractions heparin.
  • Heparin and its derivatives are capable of fix to the constituents promoting gelling and thus increasing the affinity of angiogenic factors to the constituents allowing gelation.
  • heparin binds to collagen and thus increases the affinity of bFGF for collagen.
  • the implant according to the invention can be used permanently or temporarily, for use in humans or in animals. Indeed, its ability to constitute an individualized neo-organ in vivo , allows it to be removed by sampling if necessary.
  • the subject of the invention is also a composition characterized in that it comprises a implant according to the invention and one or more others constituents such as for example an antigen, a adjuvant in particular for vaccination.
  • the means described in the invention therefore make it possible to envisage the treatment of genetic or acquired diseases over a long period, greater than several months, and this without repeated administration of the product of expression of the exogenous nucleotide sequence of which we are looking for. therapeutic activity.
  • the means described in the invention allow a quantity of expression product of the exogenous nucleotide sequence, sufficient to have a therapeutic effect in vivo , to be obtained in the form of secreted protein.
  • Another application of the recombinant cells according to the invention or of the compositions or implants containing them relates to the preparation of antibodies against the expression product of the exogenous nucleotide sequence, contained in the vector introduced into the recombinant cells.
  • these antibodies are obtained in vivo when the recombinant cells are implanted in an organism.
  • the invention allows the production in vivo of antibodies against a given antigen, for example for the purpose of vaccinating a patient.
  • Recombinant cells such as described below, or compositions or implants container can also be implemented for tumor treatment, nucleotide sequence exogenous contained in the recombinant cells encoding for a substance capable of promoting or increasing the immune response against the cells of the tumor.
  • the cells recombinants usable for the treatment of tumors may be cells obtained by recombination of tumor cells taken from a patient, with a retroviral vector responding to definitions given on the following pages.
  • the biocompatible support includes at least one of the elements chosen from group comprising PTFE, or an original support biological, particularly an original support organic based on limestone, in particular calcium carbonate, preferably coral.
  • the constituent capable of inducing and / or promoting cell gelation is preferably collagen, in particular type I collagen, preferably at a concentration of the order of 1.5 mg / ml.
  • the kit according to the invention can also include DNA comprising a sequence encoding the compound expressed and secreted by said cells. This DNA can be used to transform cells taken from the patient to be treated.
  • DNA being part of the kit is a retroviral vector as described more far.
  • Kit may also include cells having the ability to express and secrete naturally or after recombination a compound determined, for example a compound having a. interest therapeutic.
  • a provirus DNA sequence or "sequence of proviral DNA” is a DNA sequence transcribed to from the genomic RNA of the virus when it is integrated into the host cells of the virus.
  • the DNA of provirus thus comprises sequences coding for gag, pol and env proteins of the retrovirus, which correspond respectively to nucleoproteins, envelope polymerases, proteins and glycoproteins.
  • the retroviral vector according to the invention is such as the gag, pol and env genes of the provirus DNA have been deleted at least in part and this in order to obtain proviral DNA incapable of replicating, furthermore, this DNA cannot be mutated so reverse or recombine to form a wild virus.
  • the provirus DNA also contains sequences called LTR (Long Terminal Repeat), which have regions called R, U3 and U5. These LTR region sequences are involved in the cycle retrovirus replication.
  • LTR Long Terminal Repeat
  • the LTR sequence of proviral DNA also has been mutated by deletion, for example in its U3 part; this deletion affects the functions of the activator internal of the LTR region.
  • this deletion allows to decrease and preferably to neutralize the transcriptional effect of the LTR region, in retaining the capacity of the exogenous promoter of promote and control the expression of the sequence of exogenous nucleotides contained in the vector retroviral.
  • the proviral sequence upstream of the exogenous promoter is the nucleotide sequence located between nucleotides 1 and 1500 approximately depending on the numbering of the sequence shown in Figure 1.
  • This sequence upstream of the promoter is essentially devoid of all of the gag genes, pol and env of the proviral sequence.
  • exogenous nucleotide sequence is advantageously inserted under the control of the promoter exogenous, instead of the gag, pol and env sequences deleted.
  • the exogenous nucleotide sequence is advantageously inserted at the BamHI site downstream of this promoter.
  • exogenous nucleotide sequence is a sequence which is not expressed naturally in a cell in which we introduces the retroviral vector of the invention, or y is insufficiently expressed, or desired get a more important expression than the expression normal. This lack of expression or this insufficient of expression result from the nature of the cell or occurs due to a pathology affecting this cell in a given individual.
  • sequence of exogenous nucleotides code for a polypeptide determined.
  • polypeptide is meant any sequence amino acids, whatever its size, this expression including proteins and peptides.
  • the polypeptide can be in glycosylated form or not glycosylated.
  • the promoter used for the construction of recombinant retroviruses according to the invention is advantageously exogenous compared to the sequence whose he controls the expression in the sense that he is not not naturally associated. You can also use the promoter of the transferred exogenous gene.
  • the promoter used in the vector the invention may be of the constituent type or inducible. This exogenous promoter controls expression of the exogenous nucleotide sequence. Promoter may, where appropriate, be accompanied by a sequence nucleic acid regulator for example activator ("enhancer”) which would regulate its activity.
  • activator activator
  • promoters such as the mouse Mx promoter, promoters including a tetracycline operator or still promoters regulated by hormones, in especially steroids, can be used.
  • the use of internal promoters active in resting fibroblasts, such as the PGK promoter or a another housekeeping gene promoter is preferred.
  • the exogenous constitutive promoter is a promoter without TATA box and in particular the promoter of the gene for phosphoglycerate kinase (PGK-1).
  • This promoter is either the mouse promoter or the human promoter such as described by Adra et al (Gene 60, 1987, 65-74).
  • the insertion of the PGK-1 promoter into a retroviral vector allows the transfer of the gene into skin fibroblasts, for example, and expression stable at high level and very long term of this gene after fibroblasts have been implanted in vivo in a recipient, for example a mammal.
  • promoters can be used in place of the PGK-1 promoter.
  • promoters preceding the cytoskeleton genes We may also cite the promoter of ⁇ -actin. (Kort et al., 1983, Nucl. Acids Res. 11: 8287-8301) or promoter of the vimentin gene (Rettlez and Basenga (1987), Mol. Cell. Biol. 7: 1676-1685). Promoter used may not have a "TATA box".
  • a first specific retroviral vector in the context of the invention is such that a sequence of exogenous nucleotides and a constitutive promoter exogenous, as well as the proviral DNA sequence, are carried by a plasmid.
  • these sequences can be carried by the plasmid pBR322 which allows the introduction of DNA into the lines cells in which we seek to produce the vector.
  • a vector meeting the definitions is further characterized in that DNA proviral is derived from the Mo-MuLV retrovirus.
  • DNA proviral is derived from the Mo-MuLV retrovirus.
  • MuLV family retroviruses can be used and mention will be made, for example, of the HaSV or F-MuLV retroviruses.
  • the sequences of the pol and env of the proviral DNA are completely deleted.
  • the gag gene sequence may also be entirely deleted or, on the contrary, be kept in part, since the DNA of the provirus thus constituted is no longer likely to replicate.
  • a retroviral vector such as defined above is characterized in that the U3 region of the LTR3 'fragment is deleted at the level of nucleotide 2797 of Figure 1.
  • This deletion corresponds to a deletion of a fragment located between the nucleotides 7935 and 8113 according to the numbering of the sequence published by Shinnick et al (Nature 293, 543-548, 1981).
  • a retroviral vector according to the invention is the vector of type pM48 derived from Moloney virus in which the viral "enhancer" localized in V3 has been deleted and comprising an internal promoter active in fibroblasts at rest, preferably the PGK promoter or another household gene promoter, such as the vector represented in FIG. 2, modified by the nucleotide sequence exogenous at the BamHI site.
  • the retroviral vector M48 LacZ (again designated by the expression pM48 LacZ) has been deposited at the CNCM (National Culture Collection of Microorganisms, Paris - France) under N ° I-1298, the April 16, 1993.
  • This vector is derived from the vector pM48 and is characterized in that it contains downstream of the exogenous constitutive promoter, a BamHI fragment of the ⁇ -galactosidase gene.
  • This fragment of the ⁇ -galactodidase gene can easily be deleted and replaced by a sequence of exogenous nucleotides of interest, for example a sequence coding for a protein or glycoprotein likely to have a therapeutic interest or against the protein product which we would like to example get antibodies.
  • a retroviral vector of the invention can also contain a sequence capable of increasing promoter's transcriptional activity, i.e. constitutively, or inducibly.
  • the vector may contain, upstream of the exogenous promoter, an activator sequence.
  • the invention further relates to recombinant cells, characterized in that it these are cells that can be tolerated immunologically by an organism in which they would be implemented, modified by a vector retroviral meeting one of the definitions preceding.
  • Such cells can be cells from the organism in which they must be implanted after recombination by transduction with the vector of the invention, or cells lacking their surface of antigens recognized by the system immune system of the organism in which they are implanted.
  • It can also be cells endothelial, myoblast, muscle cells or irradiated or treated tumor cells according to other methods to prevent their proliferation and taken from a patient and which one wishes to modify genetic heritage so that they can intervene on the development of the tumor.
  • these recombinant cells are recombinant fibroblasts and in particular skin fibroblasts.
  • it is autologous fibroblasts versus patient in which one wishes to implant after their modification by a vector according to the invention.
  • the fibroblasts prepared from other organs can be used, such as fibroblasts isolated from an umbilical cord.
  • Fibroblasts genetically modified and used in the context of the present invention are non fibroblasts immortalized.
  • These cells can be modified by a recombinant vector encoding a polypeptide determined, said vector allowing the expression of foreign DNA in cells. They can also be modified by so-called recombination methods counterpart. Vector penetration into cells is performed using electroporation or the precipitated with calcium phosphate or by any other method involving the entry of a nucleic acid either alone or through a virus recombinant for example.
  • recombinant cells also autologous irradiated tumor cells prior to their introduction into the gel, having the ability to maintain antigens on their surface tumor accessible to the host's immune system in which the implant according to the invention was introduced.
  • the recombinant cells of the invention are also obtained either by infection of cells to be modified with a retroviral vector of the invention either by other methods of naked DNA transduction, DNA-protein complex or vector adenoviruses.
  • a retroviral vector of the invention either by other methods of naked DNA transduction, DNA-protein complex or vector adenoviruses.
  • the sequence encoding the polypeptide of which we search for the expression is introduced into proviral DNA.
  • the cells are modified by a vector containing one or more nucleotide sequences exogenous, coding for an antigen or determinant antigenic or encoding a polypeptide or a glycoprotein, soluble in serum, for example for a polypeptide or a glycoprotein of interest therapeutic, in particular for a hormone, a protein or a structural glycoprotein or a protein or a metabolism glycoprotein or a protein or a viral glycoprotein or a protein with the characteristics of an antibody or fragment antibody.
  • this sequence of exogenous nucleotides code for ⁇ -glucuronidase, or another lysosomal enzyme such as ⁇ -iduronidase or arylsulfatase B, a clotting factor such as factor VIII or factor IX, erythropoietin, or any active part of any of these proteins.
  • exogenous genetic material makes it possible to experimentally modify the properties of a tissue. Treatments based on this principle could be offered to patients with genetic deficiencies or acquired diseases.
  • the example described below uses a retroviral vector to introduce into cells the genetic information coding for a secreted protein.
  • the reimplantation in the organism of genetically modified cells is carried out by associating these cells with collagen, an angiogenic factor and the reinforcement of the coral or crosslinked collagen type.
  • the long-term in vivo expression of the genetic material thus transferred is obtained using the promoter of the murine phosphoglycerate kinase gene.
  • the retroviral vector M48 ( Figure 2) is constructed from proviral sequences isolated from genomic DNA from rat cells infected with Moloney murine leukemogenic retrovirus (Mo-MuLV).
  • Mo-MuLV Moloney murine leukemogenic retrovirus
  • the gag, pol and env genes that code for proteins viruses have been removed and replaced with sequences to convey.
  • the sequences of recombinant provirus and flanking DNA fragments cells are carried by the bacterial plasmid pBR322.
  • the mouse phosphoglycerate kinase (PGK-1) gene promoter was inserted in place of the viral genes.
  • a unique Bam HI cloning site makes it possible to place the sequences to be transported in the retroviral vector under the control of this promoter.
  • This retroviral vector has a deletion in the U3 sequences of the internal LTR (Cone et al., Mol. Cel. Biol. 7 , 887-897, 1987). This causes, in the infected cell, a strong decrease in mRNA starting at the 5 'LTR, and a predominance of mRNA produced from the promoter PGK-1. The use of this vector therefore results in the insertion into the genome of the target cell of an expression cassette for the sequence of interest under the control of the PGK-1 promoter.
  • This production is carried out by introducing the recombinant proviral structure in a line cell where the gag, pol and env genes are expressed constitutively.
  • transcomplementing or packaging line synthesizes retroviral particles lacking genomic RNA.
  • the ⁇ CRIP line derived from NIH / 3T3 mouse fibroblasts, is used here (Danos and Mulligan Proc. Natl. Acad. Sci (USA) 85 , 6460-6465, 1988).
  • the RNAs transcribed from LTR5 ′ are packaged and the cells then produce particles capable of transmitting the recombinant genome but incapable of replicating.
  • Cell culture supernatant packaging is collected after an incubation of 24 hours on the packaging cells producing the retroviral vector, and contacted with cells targets for infection.
  • a stock of around 100 ampoules containing producer cells is kept at -135 ° C. After thawing, the cells are amplified for 10 days, successively in a bottle then in rollers of 875 cm 2 , until 20 confluent rollers are obtained. Production is then started. It lasts 4 or 5 days.
  • the culture medium is DMEM containing 1 g / l of glucose, 10 mM of sodium pyruvate, and added with 5% of newborn calf serum (Hyclone). Each roller allows the packaging of 100 ml of medium per 24 hours. This culture supernatant is centrifuged to remove cell debris. The weekly production is 8 to 10 liters of viral supernatant.
  • the supernatant can be concentrated to increase the infectious titer of the retroviral vector. After centrifugation, the supernatant is dialyzed tangentially against a Sartocon membrane 100,000 porosity, using the Crossflow device of Sartorius. A concentration of 20 times is obtained in 3 hours. It is accompanied by an increase of 20 times infectious titles. After concentration, the viral preparation is used immediately, or stored at -80 ° C if the period of use is more than 2 weeks.
  • the fibroblasts intended for gene transfer are obtained by a skin biopsy carried out under very careful aseptic conditions. In mice, a syngeneic animal is sacrificed for this use. In large mammals, one or more skin fragments of approximately 10 cm 2 are removed during general anesthesia. After cutting into shreds of a few mm 2 , the fragments are dissociated by an enzymatic treatment. For a 10 cm 2 fragment, the reaction is carried out for 2 hours at 37 ° C. with gentle stirring in 50 ml of RPMI 1640 medium supplemented with 10% fetal calf serum, 100 mg of collagenase (Worthington), 500 U of dispase ( Collaborative Research Inc.).
  • the culture medium is RPMI 1640 supplemented with 10 to 20% fetal calf serum.
  • Infections with vector preparation retroviral are started the day after the implementation culture, when cell density is lower at 20% confluence. They are repeated daily, following the same protocol for 4 or 5 days. After removing the culture medium, the cells are brought into contact with the preparation of unconcentrated or concentrated retroviral vector, in all cases, added with 8 ⁇ g / ml polybrene. Incubation is carried out for 2 hours at 37 ° C, then, without prior washing, the viral preparation is replaced by culture medium.
  • the cells are amplified until a sufficient number for reimplantation is obtained, i.e. 2-6x10 8 cells / kg of body weight. Amplification is carried out in culture trays (multitray, Nunc).
  • the cells After amplification, the cells are collected by trypsin treatment. After washing and counting, they are available to be used for formation of a neo-organ. A sample is kept to control genetic transfer by Southern blot, as well as the expression and secretion of the foreign protein by an appropriate technique.
  • the whole is placed in an incubator (37 ° C, 5% CO z ) for 30 minutes in order to obtain the gelation of the collagen.
  • 1 ml of RPMI 1640 with fetal calf serum is deposited on the gel in order to compensate for variations in pH.
  • the gel is delicately detached from the edge of the culture dish using an injection needle and transferred to a larger dish, in the presence of an excess of culture medium for a period 12 to 24 hours.
  • the fibroblasts included in the collagen gel attach to the collagen fibers, which induces a retraction of the structure. It is usual to see a reduction of more than 50% in the initial volume.
  • the assembly is then ready to be implanted in the body.
  • the neo-organ is introduced into the cavity peritoneal during a laparotomy performed under General anaesthesia.
  • the implantation is performed by inserting without fixation the neo-organ between the intestinal loops in contact with the mesentery.
  • the neo-organ is fixed by two points between the two sheets of the omental apron. of the vascular connections are established from the first days.
  • the exogenous protein can then be released into circulation of the recipient animal.
  • an organ often pedunculated, encapsulated and well individualized is form. Many vascular connections are visible on its surface. There is no adhesion inflammatory. Examinations after 3 and 6 months show an identical appearance. Histological analysis at this stage reveals the presence of loose connective tissue, vascular and not very inflammatory.
  • This process was applied to the treatment of a lysosomal storage disease for which a model murine is available. It is a ⁇ -glucuronidase deficiency responsible for mucopolysaccharidosis. ⁇ -glucuronidase is phosphorylated and is therefore partly transported to the lysosomes and partly secreted into the circulation. It can be picked up by other tissues, through the receptor at mannose-6-phosphate expressed on the surface of many cells. This specific capture allows an approach therapy where the systemic distribution continues to the missing enzyme is provided by cells genetically modified.
  • 0.5 cm 3 implants containing 20 ⁇ 10 6 autologous fibroblasts are well tolerated by the mouse.
  • the implanted cells express the human ⁇ -glucuronidase gene inserted into the vector M48, under the control of the PGK promoter and secrete the foreign protein beyond 5 months.
  • the biological effect is objectified by a rapid drop in the urinary elimination of intermediate catabolites from the breakdown of mucopolysaccharides.
  • the histological normalization of the tissues of the recipient animal is spectacular, particularly in the liver and spleen. It is stable over time, and depends on the presence of the neo-organ whose excision induces a return to the initial pathological state.
  • This process is applicable to the treatment of all lysosomal storage diseases human, including Gaucher disease for which intake of a soluble form of glucocerebrosidase is possible.
  • ⁇ -L-iduronidase a lysosomal enzyme ( ⁇ -L-iduronidase) in mice with implants containing PTFE fibers, rat tail collagen and vector-modified skin fibroblasts retroviral.
  • a cDNA encoding human ⁇ -L-iduronidase was introduced into the vector M48 and a retrovirus recombinant was produced in the ⁇ CRIP line. of the nude mouse fibroblasts were placed in culture primary and infected with this retroviral vector. The cells secreting the human enzyme have been introduced in six syngeneic recipients, in two neo-organs each containing 10 million cells.
  • EPO erythropoietin
  • the nlslacZ gene was excised from the vector M48-nlslacZ by BamHI digestion and replaced by a cDNA coding for mouse EPO synthesized by PCR, thus producing the vector M48EPO.
  • a ⁇ CRE cell clone producing M48EPO retroviral particles was isolated. Fibroblasts were removed by skin biopsy from adult mice and a primary culture was established in RPMI 1640 medium with 10% FCS. These cells were infected by the M48EPO vector iteratively during the first 4 days of the culture. Analysis by upstream Southern blot that the infected cells contained on average 2 copies of the M48 EPO genome per cell. Northern blot analysis showed an expression of the cDNA of EPO very predominantly from the PGK-1 promoter.
  • a bioassay of the EPO activity secreted by these cells, as well as an ELISA test measured a secretion of 17 units of EPO per million cells per 24 hours. These cells were amplified in culture, trypsinized, then resuspended in RPMI 1640 at a concentration of 2.5 ⁇ 10 7 to 2 ⁇ 10 8 cells per ml.
  • Implants were prepared by combining the following in a well of 0.9 cm 2/1) PTFE fibers pretreated as described above to be coated successively rat tail collagen (Sigma), heparin (Roche), and bFGF (Promega); 2) 1 ml of a solution containing rat tail collagen (Sigma) at x mg / ml, bFGF (10 ⁇ g / ml) in RPMI 1640 medium; 3) a volume of 10 ⁇ l of the suspension of fibroblasts genetically modified to secrete EPO.
  • the weekly measurement of the hematocrit (normal value 46 ⁇ 1.5%) showed a gradual rise to a plateau, the level of which varied according to the number of cells secreting EPO in the implants. This plateau was 80% for 2 x 10 7 cells, 70% for 10 7 cells, 60% for 5 x 10 6 cells, 52% for 2.5 x 10 6 cells. He stayed at this level during the 6 months of animal observation.
  • the concentrations of EPO in the serum of mice carrying implants secreting EPO were 60 to 400 mU per ml (normal ⁇ 20 mU / ml).
  • Implants made of PTFE, collagen rat tail and genetically modified cells by the retroviral vector M48EPO therefore allow a long-term stable in vivo EPO secretion at high levels estimated between 500 and 1500 mU / kg / 24 hours, i.e. sufficient in humans for the correction of anemia due to hemoglobinopathies such as sickle cell anemia and ⁇ -thalassemia. DEs 10 lesser times would be sufficient for the treatment of anemia associated with chronic renal failure.
  • Biocoral® could indeed be effectively covered with different elements of the extra cellular matrix as well as angiogenic factors. It has been shown that murine type I collagen at a concentration of 0.5 mg / ml in 0.001% acetic acid adheres and covers the coral support after drying in ambient air for 12 to 24 hrs. The presence of this element of the extracellular matrix then allowed the irreversible binding of basic growth factor of fibroblasts labeled with 125 I 125 I-basic Fibroblast Growth Factor (bFGF) to Biocoral®. Fixing was thus carried out on average of 50 ng of bFGF on 40 mg of coral support pretreated with murine type I collagen.
  • bFGF Fibroblast Growth Factor
  • This rapid gesture allowed simple operative follow-ups, and suggests the possibility of implantation by laparoscopic surgery in the future. Forty five days later, an exploratory laparotomy was performed in order to practice a macroscopic assessment of the implant. Updating the omental apron was easy, without adhesion, and the location of the coral implant, containing the genetically modified autologous fibroblasts, was done very quickly. No adhesion could be noted, and the vascularization was significant, comprising large caliber vessels in connection with the implant. A process of progressive absorption of the coral has been observed for at least 4 months.
  • Biocoral is modular in its design, capable trigger sustained angiogenesis associated with its own absorption and finally devoid of systemic effects and local inflammatory type.
  • a bio-material advantageously serves as a receptacle and skeleton for a type I collagen gel containing cells genetically modified autologists capable of secrete a therapeutic factor into the circulation once the vascular connections are established.
  • EPO erythropoietin
  • mouse fibroblasts were obtained from a skin biopsy, cultured, and transduced with the vector M48EPO. After amplification, the cells were trypsinized and resuspended in RPMI 1640 medium with 10% SVFR at the concentration of 10 8 cells per ml.
  • Implants were prepared by combining the following elements in a 0.9 cm 2 well : 1) coral powder (Inoteb) formed of particles of Porosity ⁇ 45% and of particle size between 600 and 1000 ⁇ m whose pores had an average diameter of 150 ⁇ m previously treated by an incubation in a rat tail collagen solution (1 mg / ml) for 30 minutes at room temperature, dried, then incubated in a heparin solution (Roche) for 30 minutes at room temperature, dried, then incubated in a solution of bFGF (Promega) at 100 ⁇ g / ml for 30 minutes at room temperature; 2) 1 ml of a solution containing rat tail collagen (Sigma) at 3 mg / ml, bFGF (100 ⁇ g / ml) in RPMI 1640 medium; 3) a volume of 100 ⁇ l of the suspension of fibroblasts genetically modified to secrete EPO.
  • coral powder Inoteb formed of particles of Porosity ⁇ 45% and of particle size
  • Implants formed of a collagen gel structured with fragments of coral therefore allow maintenance in a functional state of genetically modified fibroblasts implanted in the peritoneal cavity of recipient animals, as well as the long-term secretion of a protein soluble in the serum.
  • EPO erythropoietin
  • Bilirubin is a breakdown product of heme produced permanently and in quantity important by the body. Bilirubin is fat-soluble, and can passively penetrate into cells, where its accumulation is toxic. The elimination of bilirubin is achieved by glucuronide conjugation in hepatocytes by bilirubin UDP-glucuronosyl transferase (bil. UDPGT) of which there are two known isoforms (1 and 2). The addition of glucuronic acid makes the molecule water-soluble, non-toxic, and allows its excretion in bile. The total hereditary deficit of activity bil.UDPGT is responsible for a very serious illness (Crigler-Najjar disease) who owes a liver transplant. There is a model of this rat disease (Gunn rat).
  • the rat cDNA coding for isoform 2 (bil.UDPGT-2) and the human cDNA coding for isoform 1 (bil.UDPGT-1) were inserted into the vector M48 after excision of the nlslacZ gene by the enzyme. BamHI, thereby generating the vectors M48GT-2 and M48GT-1.
  • Gunn rat fibroblasts were removed by a skin biopsy, placed in culture, and transduced by the vector M48GT-2. The presence of an average copy of the vector genome in transduced cells was checked by Southern blot, and expression from the PGK promoter was checked by Northern Blot.
  • the bil.UDPGT enzyme activity was detected in microsomal extracts from infected fibroblasts.
  • the cells were amplified in culture, trypsinized, then resuspended in RPMI 1640 at a concentration of 2 ⁇ 10 8 cells per ml.
  • Implants containing 2 x 10 7 genetically modified cells were prepared in the same manner as in Examples 1, 3 and 4. Two implants were inserted into the peritoneal cavity of each recipient Gunn rat. As the Gunn rat colony used is not inbred, care has been taken that each animal is reimplanted with its own cells. The measurement of unconjugated bilirubin concentrations in treated animals and in control rats of the same age showed a significant (between 20 and 50%) and stable decrease during a 2-month observation in rats carrying fibroblast implants expressing the bil.UDPGT. Bile samples were collected at the time of animal sacrifice and analyzed by high pressure liquid chromatography (HPLC).
  • HPLC high pressure liquid chromatography
  • fibroblasts of rats transduced with the vector M48UDPGT-1 and grafted in Gunn rats in the form of implants associating PTFE fibers and rat tail collagen are capable of carrying out in vivo the reaction of glucurono-conjugation of bilirubin, which conjugated bilirubin can then be eliminated in the bile by hepatocytes, thus allowing a partial correction of the phenotype of sick animals. It is therefore possible to carry out the detoxification of in vivo biological products using fibroblasts genetically modified implanted in a gel of collagen structured with PTFE fibers.
  • Mx inducible promoter
  • a retroviral vector derived of the vector M48 was constructed in which the promoter PGK-1 was excised and replaced with a fragment of 250 bp covering the -250 / +1 region of the Mx gene promoter of the mouse.
  • This promoter includes elements of response to stimulation by ⁇ and ⁇ interferons.
  • the protein Mx is expressed in response to secretion of interferon ⁇ or ⁇ .
  • the cDNA coding for EPO mouse was inserted into this vector at the BamHI site, thus generating the vector MxEPO.
  • ⁇ CRE cells producing ecotropic retroviral particles containing the genome of the vector MxEPO have been isolated. Fibroblasts isolated from a biopsy cutaneous tests performed in a mouse were infected with the vector MxEPO, and the EPO secreted in the supernatant of these cultures was measured before and after addition of interferon ⁇ (1000 units per ml) in the medium of culture. Baseline levels of EPO secretion were less than 0.3 units / ml / 24 hours reflecting a low activity of the Mx promoter present in the vector MxEPO in the absence of interferon. An increase in secretion up to 10 times the basic level has been measured after addition of ⁇ interferon. This secretion maintained for at least 6 days. This observation shows that control can be exercised over a transcriptional promoter inserted into a vector retroviral, after integrating it into the target cell genome.
  • Implants made of PTFE fibers, rat tail collagen, and containing skin fibroblasts transduced with the vector MxEPO were prepared and implanted in mice syngeneic. In the absence of other treatment, these animals maintained normal hematocrit. Some recipient animals were treated with the injection 50 mg polyA-U intraperitoneal (Boehringer) every 48 hours for two weeks to cause an elevation in hematocrit.
  • the process is applicable to distribution in the serum of any soluble protein, or truncated transmembrane proteins from their domain immobilization in the membrane. However, it does not not allow regulation of serum concentrations foreign protein.
  • the following applications are considered for genetic diseases: in hemophilia B, intake of factor IX; in hemophilia A, intake of factor VIII in the form a protein deleted in region B; in ⁇ -thalassemia, erythropoietin intake likely to correct anemia by stimulating synthesis of fetal hemoglobin.
  • soluble CD4 or its derivatives coupled with immunotoxins or immunoglobulins, is possible in retrovirus infections of the HIV family. More generally, any protein soluble antiviral could be distributed from this way.
  • the distribution of foreign protein in the mouse induces an immune response marked by the appearance of specific antibodies, as we have observed it for ⁇ -glucuronidase, CD4 soluble, and erythropoietin.
  • the process could therefore be applied to immunize animals against not yet identified product of a cDNA isolated by reverse genetics or by other means.
  • Antibodies polyclonal and monoclonal could be obtained from this way, without requiring prior preparation protein.
  • Vaccine applications could be proposed, for example, against epitopes HIV or virus envelope neutralizers related.
  • Skin fibroblasts are prepared in which we introduce two retroviral vectors, one coding for the protein of interest placed under the control of a promoter containing the elements tet operator, the other coding for the tet repressor or a related molecule modified to produce an effect activator. Depending on the nature of this last element and that of the promoter containing the operator sequences, the addition of tetracycline will induce activation or repression of transcription. Fibroblasts transduced with these two vectors are implanted in vivo in a neo-organ, and animals are treated with tetracycline to assess the induction of expression.
  • the reporter gene is preferably that of mouse EPO.
  • the progesterone receptor when it is truncated from its C-terminal region becomes capable of stimulate transcription in the presence of RU486.
  • of the skin fibroblasts are infected with two vector retrovirals, one encoding the protein of interest expressed under the control of a promoter comprising elements of response to progesterone, the other coding for a truncated version of the receiver of the progesterone.
  • fibroblasts transduced with these two vectors are implanted in neo-organs in mice, and animals are treated by RU486.

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EP02003797A 1993-04-21 1994-04-21 Biokompatibles Implantat zur in vivo Herstellung eines Neo-organs Withdrawn EP1231277A2 (de)

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FR9304700 1993-04-21
FR9304700A FR2704236B1 (fr) 1993-04-21 1993-04-21 Vecteur rétroviral pour la préparation de cellules recombinantes susceptibles d'être implantées in vivo dans un but thérapeutique.
FR9309185 1993-07-26
FR9309185A FR2708202B1 (fr) 1993-07-26 1993-07-26 Implant biocompatible pour l'expression chez l'homme ou chez l'animal de substances déterminées.
EP94914431A EP0702723B1 (de) 1993-04-21 1994-04-21 Biokompatibles implantat zur in vivo expression und sekretion von therapeutischer verbindung

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EP0702723B1 (de) 2002-09-04
ES2181717T3 (es) 2003-03-01
AU6681294A (en) 1994-11-08
US5906817A (en) 1999-05-25
EP0702723A1 (de) 1996-03-27
CA2161122A1 (fr) 1994-10-27
DE69431295T2 (de) 2003-05-15
WO1994024298A1 (fr) 1994-10-27
HK1050379A1 (zh) 2003-06-20
DK0702723T3 (da) 2003-01-13
AU688321B2 (en) 1998-03-12
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JPH08508880A (ja) 1996-09-24

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